A preparation method of myrtle ketone microcapsules

Abstract

The invention discloses a method for preparing myrtle ketone microcapsules. Myrtle is granulated after mechanical wall breaking; the granules are pre-extracted by subcritical butane to separate low polar components, and the residue is collected; the residue is leached with dichloromethane to extract myrtle ketone; the dichloromethane extraction part is subjected to vacuum rapid column chromatography to roughly separate myrtle ketone, and a mixed solvent of n-hexane and ethyl acetate is used as an eluent to collect the myrtle ketone fraction; the myrtle ketone fraction is further subdivided by vacuum rapid column chromatography, and a mixed solvent of n-hexane and dichloromethane is used as an eluent to collect the myrtle ketone fraction. The invention also discloses a method for preparing myrtle ketone microcapsules, which selects zein and maltodextrin as microcapsule wall materials, citric acid as a cross-linking agent, myrtle ketone as a core material, and food-grade monocaprylic glyceride and sucrose fatty acid ester are added as emulsifiers and plasticizers, and a spray dryer is used for spray drying to obtain myrtle ketone microcapsules.

Description

[0001] The present invention is a divisional application of the application with application number 202211313589.1, and the invention name is a method for industrial extraction of myrtle ketone and a method for preparing myrtle ketone microcapsules, and the application date is October 25, 2022. Technical Field

[0002] The invention belongs to the field of extraction of effective plant components, and particularly relates to a method for preparing myrtone microcapsules. Background Art

[0003] Myrtle (Rhodomyrtus tomentosa (Ait.) Hassk.) is a plant of the genus Myrtle in the family Myrtleaceae. It is mainly distributed in southern and southeastern China, especially in Lingnan. It is a commonly used local medicinal material in Guangdong. Myrtle is sweet and astringent in nature. Its roots, leaves, flowers and fruits are used as medicine or food. It is an economic plant with great potential for development and utilization. Previous studies have shown that myrtle fruits and leaves contain acyl phloroglucinol compounds myrtle ketone (Rhodomyrtone) with antibacterial and antiviral activities. Its molecular formula is C 26 H 34 O6, chemical name is 6,8-dihydroxy-7-(3-methylbutyryl)-9-isobutyl-2,2,4,4-tetramethyl-4,9-dihydro-1-hydrogen-oxanthene-1,3(2-hydrogen)-dione. Myrtle ketone is a fat-soluble substance, insoluble in water, soluble in organic solvents such as chloroform and methanol, and can be obtained by extraction, purification and synthesis of natural compounds. Due to the long synthesis route, low total yield and unclear residues of the current synthesis method of myrtle ketone, it is difficult to produce on a large scale. The conventional extraction method is difficult to meet the market demand due to its long extraction cycle. In addition, due to the characteristics of myrtle ketone being insoluble in water, its bioavailability is low when used directly, which limits its application in many fields. Therefore, industrial acquisition of myrtle ketone, encapsulation of myrtle ketone and improvement of its bioavailability have become the key to research. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a method for extracting myrtle ketone from myrtle and a method for preparing myrtle ketone microcapsules which are suitable for industrial implementation.

[0005] The industrial extraction method of myrtone of the present invention comprises the following steps:

[0006] (1) Myrtle is granulated after mechanical wall breaking;

[0007] (2) pre-extracting the pellets with subcritical butane to separate low-polarity components and collect the residue;

[0008] (3) extracting myrtle ketone from the residue with dichloromethane;

[0009] (4) using vacuum rapid column chromatography to crudely separate myrtle ketone from the dichloromethane extraction portion, using a mixed solvent of n-hexane and ethyl acetate as an eluent, and collecting the myrtle ketone-containing fraction;

[0010] (5) The myrtle ketone-containing fraction was further subjected to vacuum rapid column chromatography to subdivide myrtle ketone, and a mixed solvent of n-hexane and dichloromethane was used as an eluent to collect the myrtle ketone-containing fraction.

[0011] Preferably, the myrtle is dried myrtle leaves.

[0012] Preferably, the particles are pre-extracted with subcritical butane to separate low-polarity components, and the residual cake is collected by putting the particles into a subcritical extraction tank and performing continuous cyclic extraction with subcritical butane under the conditions of 0.6Mpa and 55-60°C, with a liquid-to-solid ratio of 8:1 each time, continuous extraction, releasing the extract at 60-80°C, and collecting the residual cake.

[0013] Preferably, the extraction of myrtone from the residue with dichloromethane is carried out in a material-liquid mass ratio of 10:1.

[0014] Preferably, the mixed solvent of n-hexane and ethyl acetate is a mixed solvent of n-hexane and ethyl acetate in a volume ratio of 80:20.

[0015] Preferably, the mixed solvent of n-hexane and dichloromethane is a mixed solvent of n-hexane and dichloromethane in a volume ratio of 10:90.

[0016] Preferably, the HPLC purity of the myrtone-containing fraction is greater than 80%.

[0017] The second object of the present invention is to provide a method for preparing myrtle ketone microcapsules, which comprises selecting zein and maltodextrin as microcapsule wall materials, using citric acid as a cross-linking agent, using myrtle ketone as a core material, adding food-grade monocaprylic glyceride and sucrose fatty acid ester as emulsifiers and plasticizers, and using a spray dryer for spray drying to obtain myrtle ketone microcapsules.

[0018] Preferably, the raw materials of myrtle ketone microcapsules include zein, maltodextrin, myrtle ketone, monocaprylic glyceride and sucrose fatty acid ester in a ratio of 40-57:20-3:1:26:3 by weight.

[0019] Preferably, zein and maltodextrin are dissolved in an ethanol solution to obtain a solution Sol-Z, myrtone is dissolved in ethyl acetate to obtain a solution Sol-T, monocaprylin and sucrose fatty acid ester are dissolved in water, and then the solution Sol-T and the solution Sol-Z are added, stirred and emulsified, and the ethanol and ethyl acetate are removed by heating to obtain a solvent-free zein-encapsulated myrtone core-shell structure emulsion, and then a citric acid solution is added to adjust the pH value to 3.5, and then the solution is allowed to stand to complete cross-linking, and then spray-dried to obtain myrtone microcapsules.

[0020] Preferably, the spray drying is carried out at an inlet temperature of 160-200°C, an outlet air temperature of 60-80°C, a spray pressure of 10-30 MPa, and is carried out in a constant temperature drying oven at 40-50°C.

[0021] The invention provides a method for extracting myrtle ketone from myrtle which is suitable for industrial implementation. The method can quickly and efficiently obtain high-purity myrtle ketone through mechanical wall breaking followed by granulation + subcritical butane deoiling + dichloride extraction + rapid chromatography, thereby improving an efficient extraction method for obtaining myrtle ketone.

[0022] The present invention has found that the stability of the core-shell structure microcapsules constructed by zein and maltodextrin is greatly improved through citric acid cross-linking, which is reflected in the improvement of encapsulation rate, storage stability, intestinal accessibility and other aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the extraction process flow chart of the present invention.

[0024] Figure 2 This is the HPLC chart of myrtle ketone. DETAILED DESCRIPTION

[0025] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0026] Example 1: A method for industrial extraction of high-purity myrtone, the process of which is as follows Figure 1 shown.

[0027] (1) Mechanical wall breaking of plant raw materials followed by granulation

[0028] 5550 kg of naturally sun-dried myrtle leaves were further dried in a drum airflow dryer, then ultrafinely pulverized, and then made into columnar particles using a continuous granulator to obtain 5316 kg of finished particles.

[0029] (2) Subcritical butane pre-extraction to separate low polarity components

[0030] 5316kg of granules obtained in the granulation section were put into a subcritical extraction tank and continuously circulated and extracted with subcritical butane at 0.6Mpa and 55-60℃, with a liquid-to-solid ratio of 8:1 (L:kg) for each extraction. After 7 consecutive extractions, the extract was released at 60-80℃ (heating is to improve fluidity). The subcritical extraction process obtained 132kg of myrtle subcritical extract and 5164kg of granular subcritical extraction residue. The extract contains essential oils (α-pinene, β-caryophyllene), crude fat and other low-polar substances. Some highly volatile essential oils entered the butane and were not recovered. It was determined that the extract did not contain myrtle ketone (not detected by high-performance liquid chromatography), and myrtle ketone and other high-polar substances almost all remained in the residue.

[0031] (3) Extraction of myrtle ketone with dichloromethane

[0032] Weigh 1000kg of subcritical extraction residue and put it into a 10 cubic meter room temperature extraction tank. The extraction tank is a sealed pressure vessel. 10 tons of dichloromethane are pumped into it at room temperature. The subcritical residue is extracted with a liquid-to-solid ratio of 10:1 (mass ratio). Each batch is extracted twice, and each time is 72 hours. Combine the two extraction filtrates, remove water through a molecular sieve column, and evaporate and recover dichloromethane in a single-effect evaporator to obtain 20.60kg of dichloromethane extraction concentrate with a solid content of about 50%. The sample is spin-dried and the solid content is measured to be 52.7%, and the myrone content is 1.09%. Calculated based on the average content of myrone in the subcritical residue of 0.0179%, the total extraction rate of myrone extracted by dichloromethane is 91.7%. The dichloromethane extraction process can also be carried out by other methods such as heating reflux extraction, continuous countercurrent extraction, and ultrasonic assisted extraction.

[0033] (4) Vacuum rapid column chromatography to crudely separate myrtle ketone (chromatography 1)

[0034] The dichloromethane leaching concentrate obtained in step (3) is mixed and stirred evenly with 80 mesh diatomite according to the mass ratio of 2: 1 on a 50L planetary mixer, and after the dichloromethane is recovered by decompression condensation, 21.13kg of material is obtained, which is used as a chromatography loading material for standby. On a 1# stainless steel chromatography column with an inner diameter of 1160mm and a height of 1500mm, an oil pump or a diaphragm pump is used to pump negative pressure, and 200-300 mesh silica gel is filled to a column height of 400mm (elevation), and the column layer silica gel is hammered and compacted to ensure that there are no pores, and the column layer is paved with a level to assist in paving. Between the oil pump and the chromatography column, an eluent collecting device and an eluent condensation recovery device are provided. After filling, negative pressure is continued to be maintained, and all pre-prepared diatomite chromatography loading materials (21.13kg) are weighed and evenly laid on the column layer, and a level is used to ensure paving, and then a layer of isolation cloth is laid on the sample layer to reduce the impact when the eluent is injected. An isocratic mixed solvent of normal hexane and ethyl acetate (v:v=80:20) is used as an eluent, and 0.5 column volume (about 20L) is collected each time. The components in each eluent fraction are monitored by high performance liquid chromatography. Myrtle ketone is detected in the 8th, 9th, 10th, 11th, and 12th elution fractions, among which the content of No. 10 is the highest, and the content of No. 11 and No. 12 gradually decreases. No. 13 no longer contains myrtle ketone, and elution is stopped. The elution fractions containing myrtle ketone are combined, the solvent is distilled and recovered, and the concentrate is discharged to a solid content of about 50%, and 2.47kg of concentrated solution is obtained. The sample is spin-dried, and the solid content is measured to be 45.5%, and the myrtle ketone content is 9.77%.

[0035] (5) Vacuum fast column chromatography to subdivide myrtle ketone (chromatography 2)

[0036] The myrtone concentrate obtained in step (4) is mixed with 80 mesh diatomaceous earth in a mass ratio of 2:1 on a 10L planetary mixer, and after the mixed solution of n-hexane and ethyl acetate is recovered by decompression and condensation, 2.352 kg of material is obtained as a chromatography loading material for standby use. On a 2# stainless steel chromatography column with an inner diameter of 1160 mm and a height of 1000 mm, a negative pressure is pumped by an oil pump or a diaphragm pump, and 500-800 mesh silica gel is filled to a column height of 200 mm (elevation), and the column layer silica gel is hammered and compacted to ensure that there are no pores, and the column layer is paved with a level to assist in leveling. An eluent collecting device and an eluent condensation recovery device are provided between the oil pump and the chromatography column. After filling, the negative pressure is maintained, and the diatomaceous earth chromatography loading material (2.352 kg) prepared in advance in this step is weighed and evenly laid on the column layer, and a level is used to ensure that it is leveled, and then an isolation cloth is laid flat on the sample layer to reduce the impact when the eluent is injected. A mixed solvent of n-hexane and dichloromethane (v / v=10:90) was used as the eluent, and 0.5 column volume (about 10L) was collected each time. The components in each eluent fraction were monitored by high performance liquid chromatography. Myrtle ketone was detected in the 14th, 15th, 16th and 17th elution fractions, among which the content of No. 15 was the highest, and the content of No. 16 and No. 17 decreased. No. 18 no longer contained myrtle ketone, and the elution was stopped. The elution fractions containing myrtle ketone were combined, and the solvent was distilled and recovered in a 20L rotary evaporator, evaporated to dryness, and collected by scraping bottle to obtain 116.84g of solid. The myrtle ketone content was determined by HPLC to be 85.46% ( Figure 2 ).

[0037] Example 2 Preparation of Myrtle Microcapsules by Microencapsulation Process

[0038] The microcapsule formula is shown in Table 1, and the specific steps are:

[0039] (1) Dissolve 40-57 kg of zein in 750 L of 70% ethanol, stir mechanically until completely dissolved, then add 20-3 kg of maltodextrin (the mixing ratio of zein and maltodextrin is 57:3-2:1, stir mechanically until completely dissolved, and obtain a solution Sol-Z;

[0040] (2) In another stirred container, 1 kg of myrtle ketone was dissolved in 20 L of ethyl acetate, and the solution was assisted at 50° C. for 5-10 min to obtain an ethyl acetate solution Sol-T having a myrtle ketone content of 5%;

[0041] (3) dissolving 26 kg of monocaprylin and 3 kg of sucrose fatty acid ester in water in a third stirring container, and then adding the myrtle ethyl acetate solution Sol-T obtained in step (2) and the zein solution Sol-Z obtained in step (1) simultaneously, and stirring rapidly to emulsify;

[0042] (4) while maintaining emulsification and stirring, heating to remove and recover ethanol and ethyl acetate, to obtain a solvent-free zein-encapsulated myrone core-shell structure emulsion (dispersion);

[0043] (5) adding 0-3 L of citric acid solution (100 g / L) to the dispersion obtained in step (4), and standing for 2 hours after the pH value reaches 3.5 to complete preliminary crosslinking;

[0044] (6) Use a spray dryer to spray dry, with an inlet temperature of 160-200° C., an outlet air temperature of 60-80° C., a spray pressure of 10-30 MPa, and dry in a constant temperature drying oven at 40-50° C. to obtain the final myrone microcapsule powder product.

[0045] Table 1 Myrtle ketone microcapsule formula composition

[0046]

[0047] (7) Calculation of microencapsulation efficiency EN (%)

[0048] Take a certain amount of microcapsule product, dissolve it in 70% ethanol, dissolve it completely by ultrasonication, take 2 mL and add 5 mL methanol / tetrahydrofuran (1 / 1, v / v), vortex mix and evaporate it using a vacuum concentrator. Resuspend the residue in methanol and use HPLC to detect the total content of myrone in the microcapsule product (C t );

[0049] Take a certain amount of microcapsule product, shake it in ethyl acetate, centrifuge it, take 2 mL of the supernatant, add 5 mL of methanol / tetrahydrofuran (1 / 1, v / v), install the above steps for detection, and calculate the content of free myrtone in the microcapsule product (C f );

[0050] Then the encapsulation efficiency of the microcapsules was calculated using formula (1).

[0051] EN(%)=(1-C f / C t )×100%................(1)

[0052] The encapsulation rate of 1# myrtle ketone microcapsules prepared by citric acid cross-linking and curing zein reached 91.9% (1#); in contrast, the encapsulation rate of 2# myrtle ketone microcapsules without citric acid cross-linking was only 76.7% (2#). Using 20kg maltodextrin as a filling material to replace part of zein (3#) can also successfully prepare myrtle ketone microcapsules with a higher encapsulation rate (90.2%).

[0053] Table 2 Performance test of myrtle ketone microcapsules

[0054]

[0055] (8) Calculation of bioaccessibility in the gastrointestinal environment

[0056] To simulate the gastric environment, the myrtle ketone microcapsules were added with Tris-HCl buffer (pH 3.0) and porcine pepsin (3.8 mg / mL) and incubated in a 37°C shaker (50 r / min) for 2 hours. The incubation solution was centrifuged at 15000g and 4°C for 15 minutes, 2 mL of the supernatant was added with 5 mL of methanol / tetrahydrofuran (1 / 1, v / v), vortexed and evaporated using a vacuum concentrator. The residue was resuspended in methanol and the myrtle ketone content C1 was detected by HPLC. The gastric bioaccessibility of myrtle ketone microcapsules was calculated according to formula 2.

[0057] Bioaccessibility (%) = (C 1 / 2 / C t )×100, where: C t is the total mass of myrtone in the microcapsules.............Formula 2

[0058] Intestinal environment simulation: The incubation solution obtained by simulating the gastric environment was adjusted to pH 7.0, and pig bile extract (37.8 mg / mL) and porcine pancreatic enzyme (16.25 mg / mL) were added and incubated in a shaker at 37°C for 2 h. The transparent micelle phase in the middle layer was taken and the myrtle ketone content (C2) was determined according to the above method. The gastric bioaccessibility of the myrtle ketone microcapsules was calculated according to Formula 2.

[0059] In the simulated gastric environment, the 1# microcapsules and 3# microcapsules cross-linked with citric acid were significantly more stable than the 2# microcapsules without cross-linking, which was related to the improved stability of the zein wall material after citric acid cross-linking. In the simulated intestinal environment, the 3# microcapsules showed higher bioaccessibility than the 1# microcapsules and 2# microcapsules, and the mixture of maltodextrin and zein reduced the aggregation of the microcapsules, which was helpful to improve the bioaccessibility.

Claims

1. A method for preparing myrtone microcapsules, characterized in that: Dissolving zein and maltodextrin in an ethanol solution to obtain a solution Sol-Z, dissolving myrtle ketone in ethyl acetate to obtain a solution Sol-T, dissolving monocaprylic glyceride and sucrose fatty acid ester in water, then adding the solution Sol-T and the solution Sol-Z, stirring and emulsifying, heating to remove ethanol and ethyl acetate to obtain a solvent-free zein-encapsulated myrtle ketone core-shell structure emulsion, then adding a citric acid solution to adjust the pH value to 3.5, standing to complete cross-linking, and then spray drying to obtain myrtle ketone microcapsules; The raw materials of myrtle ketone microcapsule include zein, maltodextrin, myrtle ketone, monocaprylic acid glyceride and sucrose fatty acid ester in a ratio of 40-57:20-3:1:26:3 by weight.

2. The preparation method according to claim 1, characterized in that: The spray drying is carried out in a constant temperature drying oven at 40-50°C with an inlet temperature of 160-200°C, an outlet air temperature of 60-80°C, a spray pressure of 10-30MPa.

3. The preparation method according to claim 1, characterized in that: The extraction method of myrtone comprises the following steps: (1) Myrtle is granulated after mechanical wall breaking; (2) pre-extracting the pellets with subcritical butane to separate low-polarity components and collect the residue; (3) extracting myrtle ketone from the residue with dichloromethane; (4) using vacuum rapid column chromatography to crudely separate myrtle ketone from the dichloromethane extraction portion, using a mixed solvent of n-hexane and ethyl acetate as an eluent, and collecting the myrtle ketone-containing fraction; (5) The myrtle ketone-containing fraction was further subjected to vacuum rapid column chromatography to subdivide myrtle ketone, and a mixed solvent of n-hexane and dichloromethane was used as an eluent to collect the myrtle ketone-containing fraction.

4. The preparation method according to claim 3, characterized in that: The myrtle is dried myrtle leaves.

5. The preparation method according to claim 3, characterized in that: The particles are pre-extracted with subcritical butane to separate low polar components, and the residual cake is collected by putting the particles into a subcritical extraction tank and performing continuous cyclic extraction with subcritical butane under the conditions of 0.6Mpa and 55-60°C, with a liquid-to-solid ratio of 8:1 each time, and continuous extraction is performed. The extract is released at 60-80°C and the residual cake is collected.

6. The preparation method according to claim 3, characterized in that: The residual dregs are extracted with dichloromethane to obtain myrtone in a material-liquid mass ratio of 10:

1.

7. The preparation method according to claim 3, characterized in that: The n-hexane and ethyl acetate mixed solvent is a mixed solvent of n-hexane and ethyl acetate in a volume ratio of 80:

20.

8. The preparation method according to claim 3, characterized in that: The n-hexane and dichloromethane mixed solvent is a mixed solvent of n-hexane and dichloromethane in a volume ratio of 10:90.

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